Structure-activity relationship (SAR) investigations of tetrahydroquinolines as BKCa agonists

Article abstract of DOI:10.1016/j.bmcl.2010.04.125

The membrane bound large-conductance, calcium-activated potassium channel (BKCa) is an important regulator of neuronal activity. Here we describe the identification and structure-activity relationship of a novel class of potent tetrahydroquinoline BKCa agonists. An example from this class of BKCa agonists was shown to depress the spontaneous neuronal discharges in an electrophysiological model of migraine.

Full text of DOI:10.1016/j.bmcl.2010.04.125

Bioorganic & Medicinal Chemistry Letters 20 (2010) 3573–3578
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Structure–activity relationship (SAR) investigations of tetrahydroquinolines
as BKCa agonists
a
b
b
b
a
Vijay K. Gore a, , Vu V. Ma , Ruoyuan Yin , Joe Ligutti , David Immke , Elizabeth M. Doherty ,
*
Mark H. Norman a
a Department of Chemistry Research and Discovery, Amgen Inc., One Amgen Center Drive, Thousand Oaks, CA 91320-1799, USA
b Department of Neuroscience, Amgen Inc., One Amgen Center Drive, Thousand Oaks, CA 91320-1799, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
The membrane bound large-conductance, calcium-activated potassium channel (BKCa) is an important
regulator of neuronal activity. Here we describe the identification and structure–activity relationship
of a novel class of potent tetrahydroquinoline BKCa agonists. An example from this class of BKCa agonists
was shown to depress the spontaneous neuronal discharges in an electrophysiological model of migraine.
Ó 2010 Elsevier Ltd. All rights reserved.
Received 2 March 2010
Revised 25 April 2010
Accepted 27 April 2010
Available online 20 May 2010
Keywords:
Calcium-activated potassium channel
BKCa
BKCa agonists
BKCa, the large-conductance calcium-activated potassium chan-
nel, is an important regulator of neuronal excitability and synaptic
transmission. It has been postulated that BKCa channels constantly
monitor the electrical and metabolic state of the cell based on a un-
ique activationmechanism whereby both membrane depolarization
and intracellular calcium combine to open the channel.1 Activation
of BKCa channels results in a large potassium ion efflux that causes
the cell membrane to be rapidly hyperpolarized, thereby reducing
the neuronal excitability and decreasing the intracellular calcium
load. It has been recently shown that BKCa channels are important
regulators of neuronal firing in the trigeminovascular (TNC) path-
way which is central to the pathogenesis of migraine.2 Therefore,
we sought to determine if a selective BKCa agonist could provide a
novel way to treat migraine headaches.
During the past few years, various classes of BKCa agonists (Fig. 1)
have been described, and their chemistry and pharmacology have
been reviewed.3,4 The benzimidazolone derivatives NS004 (1) and
NS1619 (2) are the prominent chemotypes among the small mole-
cule BKCa channel openers that have been studied in detail, both
in vitro and in vivo.5,6 Other class of structurally related agonists
are the 3-aryl oxindole based analogs, represented by the fluoroox-
indole Maxipost (3)7 and a series of 3-substituted-4-arylquinolin-2-
ones (4) of BKCa-channel openers have also been disclosed by
researchers at Bristol-Meyers Squibb.8
agonistic activity (2.2 0.7-fold increase in K+ current at 10
lM con-
centration) in a patch–clamp electrophysiological assay conducted
in Chinese hamster ovary (CHO) cells stably over-expressing the hu-
man BKCa channel. We initiated a structure–activity relationship
(SAR) investigation based on compound 5 to identify analogs with
improved potency and to enhance our understanding of the biolog-
ical activity of this structural class of BKCa agonists. In this paper, we
describe a SAR data set that provides insights into this novel class of
BKCa agonists.
The majority of compounds required for our SAR study were pre-
pared by the Grieco three-component cycloaddition reaction9
shown in Scheme 1. The reactions involved the condensation of
aldehydes (6), cycloalkenes (7), and anilines (8) in the presence of
trifluoroacetic acid (TFA) as the catalyst to generate the tetrahydro-
quinoline scaffold (I). The cycloaddition reaction resulted in forma-
tion of the cis–trans isomer (cis ring-fused as shown in structure I) as
the major diastereomer (>95%). The compounds initially prepared
included modifications adjacent to the tetrahydroquinoline
nitrogen (R). Accordingly, the condensations of aldehydes 6a–6p
with cyclopentadiene 7a, and aniline 8a were carried out to yield
the intermediate esters 9–24 as shown in Scheme 2. These interme-
diate esters were hydrolyzed with TFA to provide the final products
5 and 25–39.
Schemes 3–5 outline the synthesis of analogs with modifications
to the central tetrahydroquinoline core. Condensation of 2,4-dichlo-
robenzaldehyde (6m) with alkenes 7b–7d, and tert-butyl-4-amino-
benzoate (8a) were carried out to yield the intermediate esters
40–42 that were then hydrolyzed with TFA to give the final products,
43–45.
As a result of our screening efforts, we identified the tetrahydro-
quinoline 5 as a novel BKCa agonist. Compound 5 showed weak
* Corresponding author. Tel.: +1 805 447 4823; fax: +1 805 480 1337.
E-mail address: vgore@amgen.com (V.K. Gore).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.04.125

3574
V. K. Gore et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3573–3578
H
N
O
N
Cl
CF3
O
O
F
N
F3C
OH
F3C
OH
Cl
OH
HO
NH
N
H
X
MeO
O
Cl
5
1: X = Cl (NS004)
2: X = CF3 (NS1619)
3
4
MaxipostTM
Figure 1. BKCa agonists.
Additional modifications to the oxidation state of the tetrahydro-
quinoline ring system are outlined in Scheme 4. Saturated analogs
48 and 49 were prepared by the hydrogenolysis of intermediates
40 and 21 with Adam’s catalyst followed by hydrolysis with TFA.
Alternatively, compound 21 was treated with Boc-anhydride fol-
lowed by dihydroxylation using OsO4–N-methylmorpholine-N-
oxide (NMO). Subsequent deprotection of the Boc group and hydro-
lysis of the tert-butyl ester with TFA yielded the dihydroxylated ana-
log 50. The aromatization of intermediate 47 was carried out by 2,3-
dichloro-5,6-dicyanobenzoquinone (DDQ) oxidation followed by
deprotection using TFA to afford the 2,3-dihydro-1H-cyclo-
penta[c]quinoline analog 51.
densations of 2,4-dichlorobenzaldehyde (6m) with cyclopenta-
1,3-diene (7a), and anilines 8b–8e10 and were carried out to pro-
vide tetrahydroquinolines 55, 57, and 59–60. The intermediate es-
ter 55 was subjected to basic hydrolysis to give compound 56 and
lithium aluminum hydride (LAH) reduction of methyl ester 57
afforded the alcohol derivative 58.
Finally, carboxylic acid replacements 61 and 62 were synthe-
sized from compound 36 as shown in Scheme 7. Coupling of acid
36 with methoxylamine hydrochloride or methanesulfonamide in
the presence of 1-(3-dimethylaminopropyl))-3-ethylcarbodiimide
(EDC) resulted in the formation of compounds 61 and 62,
respectively.
An alternative synthetic route was required to prepare the analog
devoid of the fused cyclopentyl ring on the tetrahydroquinoline core
(Scheme 5). Methyl quinoline-6-carboxylate 52 was converted to its
corresponding N-oxide using m-CPBA followed by chlorination with
phosphorus oxychloride to yield the methyl 2-chloroquinoline-6-
carboxylate. Suzuki coupling with 2,4-dichlorophenylboronic acid
provided intermediate 53. Compound 53 was subjected to hydrog-
enolysis in the presence of Adam’s catalyst followed by basic sapon-
ification to give the final truncated product 54.
The compounds prepared in this investigation were tested for
their ability to evoke potassium current as a measure of agonism
in the PatchXpress electrophysiology assay (Molecular Devices)11
using CHO cells expressing human BKCa channel. The assay results
shown in the Tables 1–3 represent the functional activity reported
as a ratio (fold increase) of the maximum current observed com-
pared to control12 at 10 or 3
lM compound concentrations. The
fold-increase value represents the average of at least two indepen-
dent cell-recording experiments with a minimum of two cells
(n P 2) in each experiment. The higher the fold increase, the more
potent is the BKCa agonist in this system. The SAR investigations
were carried out in the three regions: A (phenyl group modifica-
tions), B (central core changes), and C (carboxylic acid replace-
ments) as highlighted in Figure 2.
Schemes 6 and 7 show the synthesis of analogs with modifica-
tions to the carboxylic acid group. Grieco three-component con-
Y
X
X
O
We first examined the effects on SAR of region A and initially
Y
+
+
evaluated the compounds at a 10 lM concentration (Table 1). Re-
R
H
H2N
R
N
H
moval of the 3-chlorophenyl group in compound 5, or replacing
it with tert-butyl group, (compounds 25 and 26, respectively) re-
sulted in complete loss of activity. The phenyl analog (27) showed
a moderate loss of potency, but the activity was retained when the
6
7
8
I
Scheme 1. General synthesis of tetrahydroquinoline scaffold (I).
O
CO2tBu
CO2tBu
CO2H
a
b
+
+
R
H
H2N
R
N
H
R
N
H
6a: R = 3-ClPh
6b: R = H
6c: R = t-Bu
9: R = 3-ClPh
10: R = H
11: R = t-Bu
5: R = 3-ClPh
25: R = H
26: R = t-Bu
7a
8a
6d: R = Ph
12: R = Ph
27: R = Ph
6e: R = 3-BrPh
6f: R = 3-MePh
6g: R = 2-ClPh
6h: R = 4-ClPh
6i: R = 4-(CF3)Ph
6j: R = 4-BrPh
6k: R = 2,3-diClPh
6l: R = 3,4-diClPh
13: R = 3-BrPh
14: R = 3-MePh
15: R = 2-ClPh
16: R = 4-ClPh
17: R = 4-(CF3)Ph
18: R = 4-BrPh
19: R = 2,3-diClPh
20: R = 3,4-diClPh
21: R = 2,4-diClPh
22: R = 2,4-diMePh
28: R = 3-BrPh
29: R = 3-MePh
30: R = 2-ClPh
31: R = 4-ClPh
32: R = 4-(CF3)Ph
33: R = 4-BrPh
34: R = 2,3-diClPh
35: R = 3,4-diClPh
36: R = 2,4-diClPh
37: R = 2,4-diMePh
6m: R = 2,4-diClPh
6n: R = 2,4-diMePh
6o: R = 3,5-dichoropyridin-2-yl
6p: R = 1-methyl-1H-pyrazol-4-yl
23: R = 3,5-dichoropyridin-2-yl
24: R = 1-methyl-1H-pyrazol-4-yl
38: R = 3,5-dichoropyridin-2-yl
39: R = 1-methyl-1H-pyrazol-4-yl
Scheme 2. Synthesis of compounds 5, 25–39. Reagents and conditions: (a) TFA, MeCN, 0 °C 8 h; (b) TFA, room temperature, 2 h, ꢀ60% (over two steps).

V. K. Gore et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3573–3578
3575
Y
Y
Cl
O
CO2tBu
CO2H
CO2tBu
Cl
Cl
a
b
Y
H
+
+
N
H
N
Cl
H2N
H
Cl
Cl
7b: Y = _CH=CH_
7c: Y = _CH2O_
7d: Y = _O_
40: Y = _CH=CH_
41: Y = _CH2O_
42: Y = _O_
43: Y = _CH=CH_
44: Y = _CH2O_
45: Y = _O_
6m
8a
Scheme 3. Synthesis of compounds 43–45. Reagents and conditions: (a) TFA, MeCN, 0 °C 8 h; (b) TFA, room temperature, 2 h, ꢀ60% (over two steps).
Z
Z
CO2tBu
Z
CO2tBu
CO2H
Cl
Cl
Cl
a
e
N
H
N
H
N
H
_
Cl
_
_
40: Z = _CH2CH2
46: Z = _CH2CH2
48: Z = _CH2CH2
Cl
Cl
_
_
_
21: Z = _CH2
47: Z = _CH2
49: Z = _CH2
HO
b, c, e
d, e
OH
CO2H
CO2H
Cl
Cl
N
H
N
51
50
Cl
Cl
Scheme 4. Synthesis of compounds 48–51. Reagents and conditions: (a) THF/EtOH, PtO2, H2, room temperature, 12 h; (b) THF, (Boc)2O, DMAP, room temperature, 12 h; (c)
cat. OsO4, NMO, aq dioxane; (d) DDQ, DCM; (e) TFA, DCM, room temperature, 2 h, 35–65%.
CO2Me
d, e
CO2H
Cl
Cl
CO2Me
a-c
N
N
H
N
53
54
52
Cl
Cl
Scheme 5. Synthesis of compound 54. Reagents and conditions: (a) m-CPBA, DCM; (b) POCl3, DCM, 18% (over two steps); (c) 2,4-dichlorophenylboronic acid, Pd(PPh3)4, DME,
1 M Na2CO3; (d) THF/MeOH, AcOH, PtO2, H2, room temperature, 12 h, 50%; (e) aq NaOH, MeOH, THF, 69% (over three steps).
Cl
O
X
X
Cl
a
H
+
+
N
H
H2N
Cl
Cl
8b: X = CH2CO2Et
8c: X = CO2Me
8d: X = CONH2
6m
7a
55: X = CH2CO2Et
b
c
56: X = CH2CO2H
57: X = CO2Me
8e: X = 1,2,4-oxadiazol-5(4
H
)-one
58: X = CH2OH
59: X = CONH2
60: X = 1,2,4-oxadiazol-5(4H)-one
Scheme 6. Synthesis of compounds 55–60. Reagents and conditions: (a) TFA, MeCN, 0 °C 8 h; (b) NaOH (1 N), EtOH, room temperature, 16 h, 67%; (c) LAH, 0 °C, THF, 49% (over
two steps).
3-chloro group was replaced with 3-bromo or 3-methyl substitu-
ents (28 and 29, respectively). On the other hand, the regio-iso-
meric analogs, 2-chlorophenyl (30) and 4-chlorophenyl (31),
showed a two- to threefold improvement in activity, clearly illus-
trating that analogs with ortho or para substituents are favored
over the meta-substituted derivatives. Replacing the 4-chloro
group with the more sterically demanding 4-trifluoromethyl (32)
or 4-bromo groups (33) showed cellular activity comparable to
31. Next we prepared a series of disubstituted analogs (34–37).
The 2,3-dichloro analog (34) did not show any improvement in
activity but the 2,4- and 3,4-disubstituted analogs (35–37) showed
a five- to sixfold enhancement in potency relative to 5. Heteroaro-
matic substitutions such as the 3,5-dichloropyridin-2-yl analog 38
and the pyrazolyl analog 39 did not provide any improvement in
the activity. Taken together these SAR results suggest that the re-
gion A substituents occupy a large hydrophobic pocket in the
receptor.
Due to the significant boost in potency obtained in the first set
of analogs, the remaining derivatives were tested at a lower con-
centration (3 lM). Based on the SAR studies for region A, we re-
tained the 2,4-dichlorophenyl group in the subsequent
compounds and examined modifications to the central core (region

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V. K. Gore et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3573–3578
O
O
O
a
Cl
N
H
N
H
61
CO2H
Cl
Cl
N
H
SO2Me
36
Cl
N
H
Cl
b
N
H
62
Cl
Scheme 7. Synthesis of compounds 61 and 62. Reagents: (a) H2NOMeÁHCl, EDC, HOBt, DIPEA, DMF, DCM, 50%; (b) EDC, DMAP, MeSO2NH2, CHCl3, 39%.
Table 1
Table 2
Structures and assay results of analogs 5 and 25–39
Structures and assay results of analogs 36, 43–45, 48–51, and 54
Cl
CO2H
Central
CO2H
Core
R
N
H
Cl
Compound Central core
Cell assaya fold increase at 3
concentration
lM
Compound
R
Cell assaya fold increase
at 10 M concentration
l
5
3-ClPh
H
t-Bu
2.2 0.7
0.6 0.2
0.8 0.1
1.2 0.2
2.6 0.1
3.1 0.9
4.9 1.6
7.3 3.9
6.0 2.9
8.9 3.3
2.7 0.5
12.1 4.9
11.2 3.5
8.9 1.2
2.5 1.4
1.0 0.3
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
36
6.7 2.9
4.5 1.4
N
H
Ph
3-BrPh
3-MePh
2-ClPh
4-ClPh
4-CF3Ph
4-BrPh
2,3-DiClPh
3,4-DiClPh
2,4-DiClPh
2,4-DiMePh
3,5-Dichloropyridin-2-yl
1-Methyl-1H-pyrazol-4-yl
43
N
H
O
44
1.5 0.2
1.0 0.3
3.6 0.8
4.6 0.6
N
H
O
a
The cellular assay was carried out in human BKCa channel expressed in CHO
cells. Results are the average of two experiments with n P 2 cells.
45
N
H
B, Table 2). Replacement of the central tetrahydro-3H-cyclo-
penta[c]quinoline core of 36 with hexahydrophenanthridine 43 re-
sulted in a moderate loss of activity. Additionally, introducing
heteroatoms and polar groups in the central core, such as com-
pounds 44, 45, and 50, resulted in a further decrease in potency,
suggesting that lipophilic functionalities in region B were preferred
for activity. On the other hand, saturating the cyclohexenyl ring to
give compound 48 did show modest activity, albeit less active than
compound 43. However, the corresponding cyclopentyl analog 49
demonstrated significant reduction in potency compared to its cor-
responding cyclopentenyl analog 36. Complete removal of the
cyclopentenyl ring to give compound 54 resulted in complete loss
of activity, as did aromatization of the core (51). These results sug-
gested that the central tetrahydro-3H-cyclopenta[c]quinoline ring
(36) was preferred.
In the next phase of our SAR investigation, we explored the ef-
fect of various carboxylic acid modifications and isosteric replace-
ments (region C, Table 3). Homologation of 36–56 was detrimental
to the activity in the cell assay. Similarly, the methyl ester (57) and
benzyl alcohol (58) analogs also showed no activity. Replacement
of the carboxylic acid with an isostere such as oxadiazolone (60)
showed moderate agonistic activity but were still weaker than its
carboxylic acid counterpart (36). The other two isosteres,
48
N
H
49
N
H
HO
OH
50
0.9 0.02
N
H
51
1.0 0.1
0.9 0.6
N
54
N
H
a
The cellular assay was carried out in human BKCa channel expressed in CHO
cells. Results are the average of two experiments with n P 2 cells.

V. K. Gore et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3573–3578
3577
acylamino methyl ether (61) and acyl sulfonamide (62) showed
weak activity.
Table 3
Structures and assay results of analogs 36, 56–58, and 60–62
Based on these SAR studies, we found that the disubstituted
aromatic groups (e.g., 2,4-dichlorophenyl) provided the optimal
substitution pattern alpha to the tetrahydroquinoline nitrogen.
Introduction of heterocyclic rings and polar groups in the central
tetrahydroquinoline ring (region B) was detrimental to the activity,
while hydrophobic groups were well tolerated in the central core
of the molecule. Replacement of the carboxylic acid group with
various isosteres did not improve the activity.
X
Cl
N
H
Cl
Compound
X
Cell assayafold increase at 3
lM concentration
36
56
57
58
CO2H
6.7 2.9
1.5 0.4
1.0 0.12
1.5 0.2
Because of its excellent in vitro potency, we evaluated whether
compound 36 would have a physiological effect on neuronal firing.
An experiment was designed to measure the spontaneous action
potentials in the rat trigeminal nucleus caudalis which is central
to the neuronal pathway of migraine. The electrophysiology-based
extracellular recording technique was used to evaluate the sponta-
neous excitability of native neurons in freshly sectioned trigeminal
nucleus caudalis slices (male, Sprague–Dawley, 3–4 weeks old,
CH2CO2H
CO2Me
CH2OH
O
N
O
60
3.4 0.04
N
H
61
62
CONHOMe
CONHSO2Me
2.2 0.6
2.7 1.5
400 lM thickness). Figure 3A illustrates an exemplary recording
of the slice electrophysiology experiment showing a dose-depen-
dent reduction in the spontaneous firing frequency elicited by
a
The cellular assay was carried out in human BKCa channel expressed in CHO
cells. Results are the average of two experiments with n P 2 cells.
the continuous perfusion of compound 36 (0.1–10
lM) into the
recording chamber. At the highest dose tested (10 M) the average
l
inhibition was ꢀ75% (Fig. 3B; n = 3 slices). The inhibitory effect of
compound 36 was reversed by co-addition of a selective BKCa
antagonist, Iberiotoxin13 (IBTX) (Fig. 3A and B), strongly suggesting
that the event was BKCa-mediated and not due to an off-target
effect.
B
C
O
OH
A
In conclusion, through SAR studies based on compound 5, we
identified a novel class of tetrahydroquinoline analogs as potent
BKCa agonists. We were able to achieve significant improvement
in the PatchXpress electrophysiology assay compared to the initial
screening-hit 5. In addition, we demonstrated that analog 36 was
Cl
N
H
Figure 2. SAR strategy for tetrahydroquinolines.
Comp-36 (µM)
0.1 0.3
Comp-36 10 µM
IBTX 0.2 µM
1
3
10
20 minutes
washing
9
6
3
0
0
2
4
6
8
10
36
38
40
Time (minutes)
100
80
60
40
20
0
**
Control
Comp-36 Comp-36+IBTX
Figure 3. Compound 36 inhibited spontaneous action potentials (SAP) in the trigeminal nucleus caudalis slices of rat. (A) A histogram, compound 36 dose-dependently
inhibited SAP. (B) Summary of the inhibition with compound 36 at 10
l
M (n = 3) on SAP and reversal of the agonism induced by compound 36 with the BKCa antagonist
**
Iberiotoxin (IBTX) (0.2
l
M) (n = 3). p = 0.01, one way ANOVA, compared with control.

3578
V. K. Gore et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3573–3578
6. Edwards, G.; Niederste-Hollenberg, A.; Schneider, J.; Noack, T.; Weston, A. H. J.
Pharmacol. 1994, 113, 1538.
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electrophysiological model of migraine.
7. Gribkoff, V. K.; Starrett, J. E., Jr.; Dworetzky, S. I.; Hewawasam, P.; Boissard, C.
G.; Cook, D. A.; Frantz, S. W.; Heman, K.; Hibbard, J. R.; Huston, K.; Johnson, G.;
Krishnan, B. S.; Kinney, G. G.; Lombardo, L. A.; Meanwell, N. A.; Molinoff, P. B.;
Myers, R. A.; Moon, S. L.; Ortiz, A.; Pajor, L.; Pieschl, R. L.; Post-Munson, D. J.;
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Acknowledgments
The authors thank their collaborators Kesheng Zhang, Alexan-
der Ernst, and their co-workers at Polyphor Ltd for their valuable
synthetic contributions to this project.
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References and notes
11. Electrophysiology assay was run using PatchXpressÒ 7000A instrument
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Sunnyvale, CA 94089-1136.
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12. Control buffer: Internal solution 70 mM KCl, 70 mM KF, 10 mM HEPES, 5 mM
HEDTA, pH 7.25; external solution 140 mM NaCl, 10 mM glucose, 10 mM
HEPES, 2 mM CaCl2, 1 mM MgCl2, pH 7.4.
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